Exposure to Titanium Dioxide Nanoparticles During Pregnancy Changed Maternal Gut Microbiota and Increased Blood Glucose of Rat
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Titanium dioxide nanoparticles (TiO2 NPs) were used worldwide for decades, and pregnant women are unable to avoid exposing to them. Studies revealed that TiO2 NPs could kill many kinds of bacteria, but whether they would affect the composition of gut microbiota, especially during pregnancy, was seldom reported. And, what adverse effects may be brought to pregnant females was also unknown. In this study, we established the prenatal exposure model of rats to explore the effects of TiO2 NPs on gut microbiota. We observed an increasing trend, but not a significant change of alpha-diversity among control and exposure groups at gestation day (GD) 10 and GD 17 during normal pregnancy process. Each different time point had unique gut microbiota operational taxonomic units (OTUs) characteristics. The abundance of Ellin6075 decreased at GD 10 and GD 17, Clostridiales increased at GD 10, and Dehalobacteriaceae decreased at GD 17 after TiO2 NPs exposure. Further phylogenetic investigation of communities by reconstruction of unobserved states (PICRUSt) prediction indicated that the type 2 diabetes mellitus related genes were enhanced, and taurine metabolism was weakened at the second-trimester. Further study showed that the rats’ fasting blood glucose levels significantly increased at GD 10 (P < 0.05) and GD 17 (P < 0.01) after exposure. Our study pointed out that TiO2 NPs induced the alteration of gut microbiota during pregnancy and increased the fasting blood glucose of pregnant rats, which might increase the potential risk of gestational diabetes of pregnant women.
KeywordsTiO2 NPs Gut microbiota Pregnancy exposure Increased fasting blood glucose
Dynamic light scattering
Linear discriminant analysis
Non-metric multi-dimensional scaling
Oral glucose tolerance test
Operational taxonomic units
Phylogenetic investigation of communities by reconstruction of unobserved states
Quantitative insights into microbial ecology
- TiO2 NPs
Titanium dioxide nanoparticles
Titanium dioxide nanoparticle (TiO2 NP) is one of the most widely used nanomaterial, and can be easily found in sunscreen, paint, ink, and foods [1, 2]. They can easily be released and enter human body during the usage of commercial products. Notably, the pregnant women cannot avoid exposing to them. Animal studies had shown that the ovarian and reproductive system dysfunction were observed , and monoaminergic neurotransmitters were also impaired  when adult female mice were exposed to TiO2 NPs. Further, pregnancy complications and adverse birth outcomes were also observed after pregnant mice exposed to TiO2 NPs . All studies above indicated that TiO2 NPs were harmful to adult female animals, as well as the pregnant females, but the mechanisms were not fully understood. So the relative studies need to be carried out for the safety evaluation of TiO2 NPs.
TiO2 NP is used as a kind of powerful antibacterial agent; they can kill many types of bacteria, including Staphylococcus aureus, Salmonella, Streptococcus mutans, and so on . The antibacterial effects were nonselective actually, while most of the current studies mainly focus on their effects on killing harmful bacteria, few reported whether TiO2 NPs would kill probiotics or other symbiotic bacteria and bring adverse effects to human beings. Studies about whether TiO2 NPs would change normal composition of gut microbiota and cause disadvantages to pregnant females were also lacking; therefore, we carried out this study from the perspective of gut microbiota.
Recently, more and more researches showed that gut microbiota were closely related with human disease including type 2 diabetes  and obesity . Probiotics could affect the metabolic of pregnant women with gestational diabetes , and change the methylation of diabetes-associated genes in fetuses . Studies reported that the plasma glucose level increased when adult mice were exposed to TiO2 NPs for 12 weeks . Whether the blood glucose of pregnant females would increase after exposure and whether the exposure period would shorten were not reported.
All the studies mentioned above suggested that TiO2 NPs may affect gut microbiota and increase the plasma glucose level, but no direct evidence proved the linkage between gut microbiota and maternal blood glucose level, and the mechanisms were also not clear. Previous studies mainly focus on adult animal studies, and the effects of TiO2 NPs on pregnant females were merely studied from the perspective of gut microbiota. In this study, we established the pregnancy exposure model of rat to explore whether the maternal gut microbiota would change and how they change after the pregnant females exposed to TiO2 NPs, and we tried to answer the issue that what adverse effects would be brought to the pregnant females by gut microbiota changes after TiO2 NPs exposure. Our study raised the concerns about the safety of TiO2 NPs to the pregnant women and we revealed the potential mechanisms.
Materials and Methods
Animal studies were performed with the permission of the ethics committee. Sprague-Dawley (SD) rats were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. Female rats (n = 8, 12 weeks old) were separated from male rats (n = 8, 14 weeks old), and rats of the same gender were kept in a large cage. All rats were housed in a temperature- (22 ± 2 °C) and humidity-controlled (40–60%) condition, with a 12 h light/dark cycle for 1 week rest. Then the female rats were randomly divided into control group (n = 4) and exposure group (n = 4), and mated with males at a 1:1 ratio in individual cages. Vaginal plug were observed every morning and the presence of vaginal plug confirmed pregnancy and recorded as gestation day 0.5 (GD 0.5), and the pregnant rats were raised in separated cages.
TiO2 NPs Preparation and Administration
TiO2 NP is a commercial product purchased from Sigma-Aldrich (13463-67-7). The stock solution of TiO2 NPs were dissolved in methylcellulose (0.5%) at the concentration of 5 mg/ml according to a previous study , and sonicated for 30 min (100 W). The hydrodynamic diameter of TiO2 NPs in methylcellulose was measured with dynamic light scattering (DLS).
Feces Collection and Fecal Total DNA Preparation
The feces of each rat were collected at GD 0 (before mating), GD 10, and GD 17 with the process of pregnancy, respectively. The feces were stored at − 80 °C before bacterial diversity were analyzed. Fecal total DNA was extracted using a Power Soil DNA kit (Mo Bio Laboratories, Carlsbad, California, USA) according to the manufacturer’s protocol. And the DNA concentrations were measured by NanoDrop spectrophotometer (NanoDrop™ 2000/2000C, USA).
16S rRNA Gene Sequencing and Data Analysis
Bacterial sequencing of 16S rRNA genes was performed with the Illumina MiSeq platform (Hangzhou Guhe Information and Technology Co., Ltd., Zhejiang, China). The V3 and V4 regions of bacterial 16S rRNA were amplified with specific primers as previously described . And the DNAs were subjected for Illumina MiSeq sequencing after amplified and purified. The sequencing data were processed using quantitative insights into microbial ecology (QIIME) according to previous studies . Data was read and merged from original DNA fragments, and the read lengths were between 400 and 500 bp. Chimeric sequences was further examined using QIIME if occurs.
Blood Sample Collection and Blood Glucose Determination
The fasting venous blood of all female rats was also collected accordingly when feces were collected. The blood samples were collected from caudal vein in the morning after 12 h of starvation at GD 0, GD 10, and GD 17, respectively. Then the fasting blood glucose levels were immediately determined with Roche ACCU-CHEK® Performa meter according to the manufacturer’s protocol after collection.
Statistical analysis were performed with Graphpad Prism 6; all data about the diversity of bacterium were presented with box plots as Mean ± SE, and significance of among all groups were examined by one-way ANOVA followed by Dunnett’s multiple comparison test. P < 0.05 was considered as statistically significant.
Results and Discussion
Characteristics of TiO2 NPs
The main characteristics of TiO2 NPs were measured and presented before animal studies. Figure 1b showed a visual field of TiO2 NPs under transmission electronic microscope. The morphology of TiO2 NPs was nearly sphere with a primary diameter of about 21 nm. The average hydrodynamic diameter was about 199.5 nm in methylcellulose solution (Fig. 1c). The purity of TiO2 NPs is ≥ 99.5%, and the surface area is 35–65 m2/g according to the manufacturer’s report. Recent studies reported that both nano- and fine grade TiO2 could increase the blood glucose level of adult animals after oral exposure [11, 16], and whether the blood glucose of the pregnant females would be affected was unknown. To make this question and underlying mechanisms clear, we established the pregnancy rat exposure model to evaluate the toxicity of TiO2 NPs and to probe the harms to pregnant rats.
Most TiO2 particles in products are with primary size of mainly ranging from 60 to 300 nm, minority (~ 20%) was < 100 nm , while recent study showed that the amount of TiO2 NPs in some food products is much larger than we known (~ 90%), for instance, chewing gum . As known, smaller nanoparticles had higher toxicity [19, 20], and the females were more sensitive to harmful substrates during pregnancy, so the minority part of TiO2 NPs may bring nonnegligible effects to pregnant females than the majority fine particles. In this study, we exposed the pregnant rat model to nanosized TiO2 (~ 21 nm) to study the potential risks of TiO2 NPs to pregnant women.
Bacteria Diversity Changes During Normal Pregnancy
Bacteria Diversity Changes After Exposed to TiO2 NPs During Pregnancy
Gut Microbiota Changes in Second Trimester After Exposed to TiO2 NPs
Gut Microbiota Changes in Late Pregnancy After Exposed to TiO2 NPs
The Effects of TiO2 NPs on Blood Glucose After Prenatal Exposure
In order to prove the results of PICRUSt prediction, we measured the rats’ fasting blood glucose at GD10 and GD17, respectively. After the pregnant rats were exposed to TiO2 NPs for 12 days (GD5–GD17), the fasting blood glucose levels were measured. As shown in Fig. 5e, comparing with control group, the rats’ fasting glucose levels increased significantly at both GD10 (P < 0.05) and GD17 (P < 0.01) after exposed to TiO2 NPs, which was in accordance with the previous reports that TiO2 NPs could increase the blood glucose level of adult animals [11, 33]. But the increment of value between control group and GD 17 was relatively small (~ 0.5 mM), and did not reach the standard of gestational diabetes . The results suggested that, maternal solitary exposed to TiO2 NPs during pregnant is not sufficient to induce gestational diabetes, but the increased blood glucose may bring adverse effects to the pregnant females and their offspring. And it was reported that maternal exposed to higher blood glucose during pregnancy might increase the risks of obesity and abnormal glucose tolerance of fetuses , which also reminded us that TiO2 NPs may bring potential risks to offspring.
Our studies indicated that prenatal exposure of TiO2 NPs could increase maternal fasting blood glucose levels, and the gut microbiota alterations might be the underlying mechanism. And we draw the conclusion that TiO2 NPs might increase the risk of gestational diabetes of human pregnant women, which should arouse our attentions.
We thank the Hangzhou Guhe Information and Technology Company for bacterial sequencing and data analysis.
This study was supported by National Natural Science Fund of China (81703256); Jiangsu Provincial Medical Youth Talent (QNRC2016307); Young Medical Talents of Changzhou (QN201504); The Open Project of The Key Laboratory of Modern Toxicology of Ministry of Education, Nanjing Medical University (NMUAMT201803); National Science Fund for Outstanding Young Scholars (81322039).
Availability of Data and Materials
The datasets used for analysis can be provided on a suitable request, by the corresponding author.
ZM designed this study and wrote the manuscript, YL and SL raised the animals and collected the testing samples, TD was responsible for the data checking and analysis, LZ contributed to figure modifying, YZ and HH helped for grammar revising and language checking, and CS helped in the linguistic errors checking and sentences modifying. All authors read and approved the final manuscript.
Ethics approval and consent to participate
This study was approved by the Ethics Committee of Nanjing Medical University, and all treatments abided by the ethics committee's standard.
The authors declare that they have no competing interests.
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
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